Method of treating influenza a

ABSTRACT

A method of treating Influenza A is disclosed. The method includes the step of administering a pharmaceutical composition including an oligonucleotide complementary to a corresponding segment of the nucleotide sequence of microRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering from Influenza A, wherein at least one of the nucleotides in the oligonucleotide is Thymidine phosphate.

FIELD OF THE INVENTION

The present invention relates to a method of treating Influenza, andmore particularly to a method of treating Influenza A.

BACKGROUND OF THE INVENTION

Influenza A virus is a virus with high morbidity, high mortality andhigh contagion. Influenza A virus can use birds or pigs as reservoirhosts in order to continuously spread and exist among different species,produce antigenic shifts through genetic reassortment, and overcome thespecies barrier to infect humans. Moreover, antigenic drifts caused byquick and unpredictable mutations of the influenza A virus causes peoplewho have been infected with the influenza A virus to be infected again.Because of these two antigenic evolutionary mechanisms, an epidemic orpandemic influenza occurs every few years or decades. Millions of peopleare infected every year, causing a tremendous burden to health and theeconomy.

A microRNA (abbreviated miRNA) is a small RNA molecule containing about22 nucleotides. After transcription, a primary miRNA (pri-miRNA) iscleaved by RNase III enzyme Drosha in the nucleus, and RNase III enzymeDrosha can cleave the double stranded pri-miRNA near the stem-loop andproduce a precursor-miRNA (pre-miRNA) having a stem-loop structure and60-70 nucleotides. Pre-miRNA is exported out of the nucleus byExportin-5. In the cytoplasm, the pre-miRNA hairpin is cleaved by theRNase III enzyme Dicer, yielding a miRNA:miRNA* duplex about 22nucleotides in length, and one strand of the miRNA:miRNA* duplex isincorporated into the RNA-induced silencing complex (RISC). The functionof the RISC is protein translation inhibition.

In recent years, substantial literature has indicated that miRNA canregulate viral replication. However, whether there exists aspecies-specific miRNA which can regulate influenza A viral replicationremains unknown.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, a method ofdiagnosing Influenza A is disclosed. The method includes the step ofdetermining a level of microRNA-1290 (miR-1290) in a cell of a subjectbased on a measurement of a level of an oligonucleotide complementary toa corresponding segment of the nucleotide sequence of miR-1290 (SEQ IDNO: 1), wherein at least one of the nucleotides in the oligonucleotideis Thymidine phosphate.

In accordance with another aspect of the present invention, a method oftreating Influenza A is disclosed. The method includes the step ofadministering a pharmaceutical composition including an oligonucleotidecomplementary to a corresponding segment of the nucleotide sequence ofmicroRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering fromInfluenza A, wherein at least one of the nucleotides in theoligonucleotide is Thymidine phosphate.

In accordance with a further aspect of the present invention, a methodof treating Influenza A is disclosed. The method includes the step ofadministering a pharmaceutical composition including an oligonucleotidecomplementary to a corresponding segment of the nucleotide sequence ofmicroRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering fromInfluenza A.

The above objects and advantages of the present invention will becomemore readily apparent to those ordinarily skilled in the art afterreviewing the following detailed descriptions and accompanying drawings,in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) shows the Ct-number equation obtained by real-time polymerasechain reaction (real-time PCR) through serial dilution of pre-miR-1290(Ambion, Pre-miR™ miRNA Starter Kit);

FIG. 1(b) shows the verification of expression levels of the screenedmiRNA Homo sapiens miRNA-1290 (hsa-miR-1290) after A/WSN/33 (H1N1)multiple infection by real-time polymerase chain reaction (real-timePCR);

FIG. 1(c) shows the verification of expression levels of the screenedmiRNA hsa-miR-1290 after A/WSN/33 (H1N1) single infection by real-timepolymerase chain reaction (real-time PCR);

FIG. 2(a) shows the transfection efficiency of hsa-miR-1290 in A549cells;

FIG. 2(b) shows the viral protein expression in A549 cells transfectedwith hsa-miR-1290 after A/WSN/33 (H1N1) multiple infection;

FIG. 2(c) shows the viral production of a single cell transfected withhsa-miR-1290 after A/WSN/33 (H1N1) multiple infection;

FIG. 3(a) shows the transfection efficiency of hsa-miR-1290 in A549cells;

FIG. 3(b) shows the viral protein expression in A549 cells transfectedwith hsa-miR-1290 after A/WSN/33 (H1N1) single infection;

FIG. 3(c) shows the viral production of a single cell transfected withhsa-miR-1290 after A/WSN/33 (H1N1) single infection;

FIG. 4(a) shows the transfection efficiency of locked nucleic acid miRNA1290 (LNA-1290) in A549 cells;

FIG. 4(b) shows the viral protein expression in A549 cells transfectedwith LNA-1290 after A/WSN/33 (H1N1) multiple infection;

FIG. 4(c) shows the viral production of a single cell transfected withLNA-1290 after A/WSN/33 (H1N1) multiple infection;

FIG. 5(a) shows the transfection efficiency of hsa-miR-1290 in A549cells;

FIG. 5(b) shows the viral protein cAMP responsive element bindingprotein 3-like 2 (CREB3L2) expression in A549 cells transfected withhsa-miR-1290;

FIG. 5(c) shows the viral protein polymerase I and transcript releasefactor (PTRF) expression in A549 cells transfected with hsa-miR-1290;

FIG. 5(d) shows the viral protein Vimentin (VIM) expression in A549cells transfected with hsa-miR-1290;

FIG. 5(e) shows the Vimentin mRNA expression in A549 cells transfectedwith hsa-miR-1290;

FIG. 6(a) shows the transfection efficiency of LNA-1290 in A549 cells;

FIG. 6(b) shows human Vimentin-3′untranslated region (hVIM-3′UTR)expression in A549 cells transfected with LNA-1290;

FIG. 7(a) shows the inhibition efficiency of Vimentin in A549 cellstransfected with siVIM;

FIG. 7(b) shows the viral protein expression in A549 cells transfectedwith siVIM;

FIG. 7(c) shows the viral production of a single cell transfected withsiVIM; and

FIG. 8 shows human Vimentin-3′untranslated region (hVIM-3′UTR) and avianVimentin-3′ untranslated region (aVIM-3′UTR) expressions after A/WSN/33(H1N1) infection in a single replication cycle.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more specifically withreference to the following embodiments. It is to be noted that thefollowing descriptions of preferred embodiments of this invention arepresented herein for the purposes of illustration and description only;they are not intended to be exhaustive or to be limited to the preciseform disclosed.

According to an embodiment of the present invention, a method ofdiagnosing Influenza A is disclosed. The method includes the step ofdetermining a level of microRNA-1290 (miR-1290) in a cell of a subjectbased on a measurement of a level of an oligonucleotide complementary toa corresponding segment of the nucleotide sequence of miR-1290 (SEQ IDNO: 1), wherein at least one of the nucleotides in the oligonucleotideis Thymidine phosphate.

The virus subtypes of Influenza A may be H7N9, H1N1, H5N1, H1N2, H2N2,H2N3, H3N1, H3N2, H3N8, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4,N7N7, H9N2, H10N7 or H10N8, preferably H1N1. The subject may be a human.

The oligonucleotide complementary to the corresponding segment of thenucleotide sequence of miR-1290 may be cDNA. The cDNA may be produced byreverse transcription using the miR-1290 as a template. The method mayfurther include the step of amplifying the cDNA of the miR-1290 usingpolymerase chain reaction (PCR). The PCR may be real-time polymerasechain reaction (real-time PCR). The corresponding segment of thenucleotide sequence of miR-1290 may be the total or a part of SEQ IDNO: 1. The level of the oligonucleotide is proportional to the level ofmicroRNA-1290. When the subject is infected with Influenza A, the levelof microRNA-1290 significantly increases and consequently the level ofthe oligonucleotide also significantly increases. Thus, when asignificant increase in the level of the oligonucleotide is measured, itcan be diagnosed that the subject is infected with Influenza A.

According to an embodiment of the present invention, a method oftreating Influenza A is disclosed. The method includes the step ofadministering a pharmaceutical composition including an oligonucleotidecomplementary to a corresponding segment of the nucleotide sequence ofmicroRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering fromInfluenza A, wherein at least one of the nucleotides in theoligonucleotide is Thymidine phosphate.

The pharmaceutical composition may be powder or liquid, and thepharmaceutical composition may be administered nasally. Some other miRNAinhibitors, such as miR-122 inhibitor, are administered via vascularinjection and are thus easy to degrade. The pharmaceutical compositionin the present invention has no such problem because it is administerednasally.

Today, most drugs for treating Influenza are against viral proteins.Viral proteins are prone to mutate, and thus it is easy for them toacquire drug resistance. The pharmaceutical composition in the presentinvention is against host cells. Host cells do not mutate easily, andthus the pharmaceutical composition in the present invention is unlikelyto produce drug resistance.

The virus subtypes of Influenza A may be H7N9, H1N1, H5N1, H1N2, H2N2,H2N3, H3N1, H3N2, H3N8, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4,N7N7, H9N2, H10N7 or H10N8, preferably H1N1. The subject may be a human.

The pharmaceutical composition can restore the expression of Vimentin,which is inhibited by miR-1290.

The nucleotides in the oligonucleotide may be independently selectedfrom the group consisting of a DNA unit, an RNA unit, a LNA (lockednucleic acid) unit, a 2′-OMe DNA unit and a 2′-OMe RNA unit. At leastone of the internucleoside linkages between the nucleotides in theoligonucleotide may be a phosphorothioate internucleoside linkage. Theoligonucleotide may be 5′- or 3′-cholesteryl-oligonucleotide.Preferably, the oligonucleotide has the nucleotide sequence of SEQ IDNO: 2.

According to an embodiment of the present invention, a method oftreating Influenza A is disclosed. The method includes the step ofadministering a pharmaceutical composition including an oligonucleotidecomplementary to a corresponding segment of the nucleotide sequence ofmicroRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering fromInfluenza A.

At least one of the nucleotides in the oligonucleotide is chemicallymodified. Chemical modification can make the oligonucleotide stable.

The pharmaceutical composition may be powder or liquid, and thepharmaceutical composition may be administered nasally.

The virus subtypes of Influenza A may be H7N9, H1N1, H5N1, H1N2, H2N2,H2N3, H3N1, H3N2, H3N8, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4,N7N7, H9N2, H10N7 or H10N8, preferably H1N1. The subject may be a human.

The pharmaceutical composition can restore the expression of Vimentin,which is inhibited by miR-1290.

The nucleotides in the oligonucleotide may be independently selectedfrom the group consisting of a DNA unit, an RNA unit, a LNA (lockednucleic acid) unit, a 2′-OMe DNA unit and a 2′-OMe RNA unit. At leastone of the internucleoside linkages between the nucleotides in theoligonucleotide may be a phosphorothioate internucleoside linkage. Theoligonucleotide may be 5′- or 3′-cholesteryl-oligonucleotide.Preferably, the oligonucleotide has the nucleotide sequence of SEQ IDNO: 2.

Experiments 1. Analyzing miRNA Expressions in A549 and DF-1 Cells afterA/WSN/33 (H1N1) Infection by Microarray

Infect A549 and DF-1 cells with 0.001 MOI (multiplicity of infection)A/WSN/33 (H1N1) for 12, 24 and 36 hours or 2 MOI A/WSN/33 (H1N1) for 2,6 and 10 hours, and then analyze miRNA expressions in A549 and DF-1cells by microarray. As a result, both miRNA expressions in A549 andDF-1 cells are influenced by A/WSN/33 (H1N1) infection. Find theintersections of miRNAs whose expression levels, before and afterinfection, have a difference over 1.5 times and miRNAs having nohomology with birds. As a result, 8 miRNAs and 12 miRNAs are screenedwith 0.001 MOI and 2 MOI.

2. Verifying Expression Levels of the Screened miRNAs after A/WSN/33(H1N1) Infection by Real-Time Polymerase Chain Reaction (Real-Time PCR)

Infect A549 cells with 0.001 MOI or 2 MOI A/WSN/33 (H1N1), and extracttotal RNAs of A549 cells with Trizol (Invitrogen) for 12, 24 and 36hours or 2, 6 and 10 hours, respectively. Take 500 ng of total RNA andsynthesize cDNA with Superscript III Reverse Transcriptase (Invitrogen),take 1 μl cDNA to mix with SYBR Green Master Mix (KAPA), react at 16° C.for 30 minutes, react at 20° C. for 30 seconds, react at 42° C. for 30seconds and react at 50° C. for 1 second, and then repeat 50 times toamplify the templates. Input the obtained Ct value into the Ct-numberequation obtained by serial dilution of pre-miR-1290 (Ambion, Pre-miR™miRNA Starter Kit), as shown in FIG. 1(a), so as to obtain the copynumber of Homo sapiens miRNA-1290 (hsa-miR-1290). The results show thathsa-miR-1290 has 5.5 and 1.9 times differences after 24 and 2 hoursinfection with 0.001 MOI and 2 MOI, respectively, as shown in FIG. 1(b)and FIG. 1(c).

3. Hsa-miR-1290 Expression in A549 Cells Increases Viral Production ofA/WSN/33 (H1N1) in Multiple Replication Cycles

Transfect 50 nM pre-miR negative control or 50 nM pre-miR-1290 into A549cells with Lipofectamine RNAiMAX (Introgen), collect cells with Trypsinafter one day, seed cells in a 12 well plate, and infect cells with0.001 MOI A/WSN/33 (H1N1) for 24, 48 and 72 hours after 24 hours.Extract total RNAs on the fifth day, and analyze the transfectionefficiency of hsa-miR-1290 by real-time PCR and inner standardizing datawith U6, as shown in FIG. 2(a). Collect cells and supernatant atdifferent infection time points. Analyze viral proteins with Westernblot and using β-actin as the inner standard. Analyze viral expressionwith plaque assay. The results show that the expression of hsa-miR-1290significantly increases the expression of viral proteins 48 and 72 hoursafter infection, as shown in FIG. 2(b). Viral production of a singlecell transfected with hsa-miR-1290 is ten times larger than that of thenegative control, as shown in FIG. 2(c).

4. Hsa-miR-1290 Expression in A549 Cells Increases Viral Production ofA/WSN/33 (H1N1) in a Single Replication Cycle

Transfect 50 nM pre-miR negative control or 50 nM pre-miR-1290 into A549cells with Lipofectamine RNAiMAX (Introgen), collect cells with Trypsinafter one day, seed cells in a 12 well plate, and infect cells with 2MOI A/WSN/33 (H1N1) for 2, 6 and 10 hours after 24 hours. Extract totalRNAs on the 58^(th) day, and analyze the transfection efficiency ofhsa-miR-1290 by real-time PCR and inner standardizing data with U6, asshown in FIG. 3(a). Collect cells and supernatant at different infectiontime points. Analyze viral proteins with Western blot and using β-actinas the inner standard. Analyze viral expression with plaque assay. Theresults show that the expression of hsa-miR-1290 significantly increasesthe expression of viral proteins 10 hours after infection, as shown inFIG. 3(b). Viral production of a single cell transfected withhsa-miR-1290 also increases significantly 10 hours after infection, asshown in FIG. 3(c).

5. Locked Nucleic Acid miRNA 1290 (LNA-1290) Expression in A549 CellsIncreases Viral Production of A/WSN/33 (H1N1) in Multiple ReplicationCycles

Transfect 50 nM LNA (locked nucleic acid) negative control (EXIQON) or25 nM locked nucleic acid miRNA 1290 (LNA-1290) (EXIQON) into A549 cellswith Lipofectamine RNAiMAX (Introgen), collect cells with Trypsin afterone day, seed cells in a 12 well plate, and infect cells with 0.001 MOIA/WSN/33 (H1N1) for 24, 48 and 72 hours after 24 hours. Extract totalRNAs on the fifth day, and analyze the transfection efficiency ofLNA-1290 by real-time PCR and inner standardizing data with U6, as shownin FIG. 4(a). Collect cells and supernatant at different infection timepoints. Analyze viral proteins with Western blot and using β-actin asthe inner standard. Analyze viral expression with plaque assay. Theresults show that compared to the negative control, the expression ofLNA-1290 significantly decreases the expression of viral proteins 48 and72 hours after infection, as shown in FIG. 4(b). Viral production alsodecreases by at least ten times after inhibiting the expression ofhsa-miR-1290, as shown in FIG. 4(c).

6. Hsa-miR-1290 Expression in A549 Cells Inhibits Endogenous VimentinExpression

Use TargetScan and miRanda to predict target genes of hsa-miR-1290. As aresult, cAMP responsive element binding protein 3-like 2 (CREB3L2),polymerase I and transcript release factor (PTRF) and Vimentin (VIM) arepredicted. Transfect 50 nM pre-miR negative control or 50 nMpre-miR-1290 into A549 cells with Lipofectamine RNAiMAX (Introgen) for72 hours. Extract total RNAs and proteins. Analyze the transfectionefficiency of hsa-miR-1290 by real-time PCR and inner standardizing datawith U6, as shown in FIG. 5(a). Take 30 μg total proteins to analyzetarget genes expression by Western blot and inner standardizing datawith β-actin. Divide the value obtained by transfecting pre-miR-1290 bythe value obtained by transfecting pre-miR negative control to obtain aratio. Vimentin protein expression is suppressed to 40% by hsa-miR-1290,but CREB3L2 and PTRF protein expressions do not change significantly, asshown in FIGS. 5(b)-5(d). Vimentin mRNA expression is also suppressed to30% by hsa-miR-1290, as shown in FIG. 5(e). The results show thathsa-miR-1290 can inhibit Vimentin expression by degrading mRNA.

7. LNA-1290 Expression in A549 Cells Restores HumanVimentin-3′Untranslated Region (hVIM-3′UTR) Degradation

Transfect pMIR-Fluc-hVIM-3′UTR and pRL-TK into A549 cells for 12 hours,and then transfect 25 nM LNA-1290 into A549 cells for 12 hours. Infectcells with 2 MOI A/WSN/33 (H1N1) for 6 hours. Extract total RNA, andanalyze the transfection efficiency of LNA-1290 by real-time PCR andinner standardizing data with U6, as shown in FIG. 6(a). Analyzeexpression levels of Firefly luciferase (Fluc) and Renilla luciferase(Rluc) with dual-luciferase reporter assay (Promega) to obtain aFluc/Rluc value. As a result, the expression of humanVimentin-3′untranslated region (hVIM-3′UTR) decreases by about 25% afterinfecting cells with A/WSN/33 (H1N1). However, if LNA-1290 is expressedbefore the infection, the suppressed hVIM-3′UTR is restored, as shown inFIG. 6(b). The result shows the inhibition specificity of hsa-miR-1290to Vimentin.

8. Inhibiting Vimentin Expression Increases Viral Production of A/WSN/33(H1N1) in A549 Cells in Multiple Replication Cycles

Transfect 60 pmole siVIM (Ambion) or Scrl (GeneDirex) into A549 cellswith Lipofectamine RNAiMAX (Introgen), collect cells with Trypsin after24 hours, seed cells in a 12 well plate, and infect cells with 0.001 MOIA/WSN/33 (H1N1) for 24, 48 and 72 hours after 24 hours. Extract totalRNAs on the fifth day, and take 30 μg total proteins to monitor theinhibition efficiency of Vimentin, as shown in FIG. 7(a). Analyze viralproteins with Western blot and using β-actin as the inner standard.Analyze viral expression with plaque assay. As a result, compared to thecontrol, viral proteins increase significantly 72 hours after infectionwhen inhibiting Vimentin expression, and viral expression levelincreases 10 times 72 hours after infection, as shown in FIGS.7(b)-7(c). Inhibiting Vimentin expression and expressing has-miR-1290both positively regulate A/WSN/33 (H1N1) replication. This further showsthat Vimentin and has-miR-1290 regulate A/WSN/33 (H1N1) on the samepathway.

9. Human Vimentin-3′Untranslated Region (hVIM-3′UTR) and AvianVimentin-3′Untranslated Region (aVIM-3′UTR) Expressions after A/WSN/33(H1N1) Infection in a Single Replication Cycle

Co-transfect pMIR-hVIM-3′UTR-Fluc or pMIR-aVIM-3′UTR-Fluc into A549 orDF-1 cells for 24 hours, and infect cells with 2 MOI A/WSN/33 (H1N1) for2, 6 and 10 hours. Analyze expression levels of Firefly luciferase(Fluc) and Renilla luciferase (Rluc) with dual-luciferase reporter assay(Promega) to obtain a Fluc/Rluc value. After obtaining the Fluc/Rlucvalue, divide the Fluc/Rluc value obtained by an infected sample by theFluc/Rluc value obtained by an uninfected sample. The results show thathVIM-3′UTR expression in A549 cells decreases to 60%-80% afterinfection. aVIM-3′UTR expression in DF-1 cells increases to 120%-140%after infection, as shown in FIG. 8. A/WSN/33 (H1N1) indeed onlyinhibits Vimentin expression in A549 cells, and regulates Vimentinexpression with species specificity.

Embodiments

1. A method of diagnosing Influenza A, comprising the step ofdetermining a level of microRNA-1290 (miR-1290) in a cell of a subjectbased on a measurement of a level of an oligonucleotide complementary toa corresponding segment of the nucleotide sequence of miR-1290 (SEQ IDNO: 1), wherein at least one of the nucleotides in the oligonucleotideis Thymidine phosphate.2. The method of Embodiment 1, wherein the oligonucleotide is cDNAproduced by reverse transcription using the miR-1290 as a template.3. The method of Embodiments 1-2, further comprising the step ofamplifying the cDNA of the miR-1290 using polymerase chain reaction(PCR).4. The method of Embodiments 1-3, wherein the Influenza A is caused by aH1N1 virus.5. The method of Embodiments 1-4, wherein the subject is a human.6. A method of treating Influenza A, comprising the step ofadministering a pharmaceutical composition including an oligonucleotidecomplementary to a corresponding segment of the nucleotide sequence ofmicroRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering fromInfluenza A, wherein at least one of the nucleotides in theoligonucleotide is Thymidine phosphate.7. The method of Embodiment 6, wherein the pharmaceutical composition isadministered nasally.8. The method of Embodiments 6-7, wherein the pharmaceutical compositionreduces H1N1 viral production.9. The method of Embodiments 6-8, wherein the pharmaceutical compositionrestores the expression of Vimentin.10. The method of Embodiments 6-9, wherein the subject is a human.11. The method of Embodiments 6-10, wherein the nucleotides in theoligonucleotide are independently selected from the group consisting ofa DNA unit, an RNA unit, a LNA (locked nucleic acid) unit, a 2′-OMe DNAunit and a 2′-OMe RNA unit.12. The method of Embodiments 6-11, wherein at least one of theinternucleoside linkages between the nucleotides in the oligonucleotideis a phosphorothioate internucleoside linkage.13. The method of Embodiments 6-12, wherein the oligonucleotide is 5′-or 3′-cholesteryl-oligonucleotide.14. The method of Embodiments 6-13, wherein the oligonucleotide has thenucleotide sequence of SEQ ID NO: 2.15. A method of treating Influenza A, comprising the step ofadministering a pharmaceutical composition including an oligonucleotidecomplementary to a corresponding segment of the nucleotide sequence ofmicroRNA-1290 (miR-1290) (SEQ ID NO: 1) to a subject suffering fromInfluenza A.16. The method of Embodiment 15, wherein at least one of the nucleotidesin the oligonucleotide is chemically modified.17. The method of Embodiments 15-16, wherein the pharmaceuticalcomposition reduces H1N1 viral production.18. The method of Embodiment 15-17, wherein the pharmaceuticalcomposition restores the expression of Vimentin.19. The method of Embodiment 15-18, wherein the nucleotides in theoligonucleotide are independently selected from the group consisting ofa DNA unit, an RNA unit, a LNA (locked nucleic acid) unit, a 2′-OMe DNAunit and a 2′-OMe RNA unit.20. The method of Embodiment 15-19, wherein at least one of theinternucleoside linkages between the nucleotides in the oligonucleotideis a phosphorothioate internucleoside linkage.

1. A method of diagnosing Influenza A, comprising the step ofdetermining a level of microRNA-1290 (miR-1290) in a cell of a subjectbased on a measurement of a level of an oligonucleotide complementary toa corresponding segment of the nucleotide sequence of miR-1290 (SEQ IDNO: 1), wherein at least one of the nucleotides in the oligonucleotideis Thymidine phosphate.
 2. The method according to claim 1, wherein theoligonucleotide is cDNA produced by reverse transcription using themiR-1290 as a template.
 3. The method according to claim 2, furthercomprising the step of amplifying the cDNA of the miR-1290 usingpolymerase chain reaction (PCR).
 4. The method according to claim 1,wherein the Influenza A is caused by a H1N1 virus.
 5. The methodaccording to claim 1, wherein the subject is a human.
 6. A method oftreating Influenza A, comprising the step of administering apharmaceutical composition including an oligonucleotide complementary toa corresponding segment of the nucleotide sequence of microRNA-1290(miR-1290) (SEQ ID NO: 1) to a subject suffering from Influenza A,wherein at least one of the nucleotides in the oligonucleotide isThymidine phosphate.
 7. The method according to claim 6, wherein thepharmaceutical composition is administered nasally.
 8. The methodaccording to claim 6, wherein the subject is suffering from H1N1 and thepharmaceutical composition reduces H1N1 viral production.
 9. The methodaccording to claim 6, wherein the pharmaceutical composition restoresthe expression of Vimentin.
 10. The method according to claim 6, whereinthe subject is a human.
 11. The method according to claim 6, wherein thenucleotides in the oligonucleotide are independently selected from thegroup consisting of a DNA unit, an RNA unit, a LNA (locked nucleic acid)unit, a 2′-OMe DNA unit and a 2′-OMe RNA unit.
 12. The method accordingto claim 6, wherein at least one of the internucleoside linkages betweenthe nucleotides in the oligonucleotide is a phosphorothioateinternucleoside linkage.
 13. The method according to claim 6, whereinthe oligonucleotide is 5′- or 3′-cholesteryl-oligonucleotide.
 14. Themethod according to claim 6, wherein the oligonucleotide has thenucleotide sequence of SEQ ID NO:
 2. 15. A method of treating InfluenzaA, comprising the step of administering a pharmaceutical compositionincluding an oligonucleotide complementary to a corresponding segment ofthe nucleotide sequence of microRNA-1290 (miR-1290) (SEQ ID NO: 1) to asubject suffering from Influenza A.
 16. The method according to claim15, wherein at least one of the nucleotides in the oligonucleotide ischemically modified.
 17. The method according to claim 15, wherein thesubject is suffering from H1N1 and the pharmaceutical compositionreduces H1N1 viral production.
 18. The method according to claim 15,wherein the pharmaceutical composition restores the expression ofVimentin.
 19. The method according to claim 15, wherein the nucleotidesin the oligonucleotide are independently selected from the groupconsisting of a DNA unit, an RNA unit, a LNA (locked nucleic acid) unit,a 2′-OMe DNA unit and a 2′-OMe RNA unit.
 20. The method according toclaim 15, wherein at least one of the internucleoside linkages betweenthe nucleotides in the oligonucleotide is a phosphorothioateinternucleoside linkage.